Nuclear magnetic resonance (NMR) spectroscopy continues to play a central role in the characterization of the structure and dynamics of proteins, nucleic acids, carbohydrates and their complexes. Over the past fifteen years there have been staggering developments in NMR techniques and supporting technologies such that the comprehensive structural characterization of 20 kDa proteins is becoming almost routine. Second only to crystallography, NMR spectroscopy provides an unparalleled view of structure and it remains second to none in its ability to examine dynamic phenomena. NMR also provides a unique avenue to monitor the full structural and dynamic effects of changes in temperature, solution conditions and the binding of small and large ligands.
Application of NMR to Analysis of Protein Structure
The size of proteins that can be analyzed by modern NMR techniques has dramatically increased over the past decade. Coupled with the introduction of heteronuclear (Sorensen et al., 1987, Progr. NMR Spectroscopy 16:163-192.) and ultimately triple resonance (Kay et al., 1990, J. Magn. Reson. 89:495-514.) spectroscopy, was the widespread use of recombinant technologies to introduce NMR-active isotopes into proteins and nucleic acids (LeMaster, 1994 Progr. NMR Spectroscopy 26:371-419; McIntosh & Dahlquist, 1990, Q. Rev. Biophys. 23:1-38). With the development of multinuclear and multidimensional capabilities, NMR is now able to confidently, efficiently and comprehensively deal with small proteins with significant spectral complexity. However, for large proteins, increasing size brings with it several important limitations which unfortunately compound each other. This severely limits the size of a protein that can be efficiently approached by modern NMR techniques.
One limitation is that increasing size leads to slower tumbling and correspondingly shorter spin-spin relaxation times. The basic engine of NMR spectroscopy of proteins, the triple resonance technology, begins to fail. As lines broaden, basic sensitivity also becomes a limiting issue. Another limitation is that increasing size leads to increasingly complex spectra: Spectral degeneracy complicates the assignment process and renders assignment of NOEs to parent hydrogens problematic.
One way to reduce the problems posed by the size and hence complexity of the protein is to reduce the limitations presented by short spin-spin relaxation times. As already mentioned, increasing size leads to shorter spin-spin relaxation times. Since the coherence transfer processes underlying current triple resonance-based assignment strategies are time-dependent, these approaches begin to fail with proteins .about.30 kDa and larger. Random partial or perdeuteration has been used to successfully reduce the dipolar field such that high resolution .sup.15 N-HSQC spectra can be obtained (LeMaster, 1994, supra). Unfortunately, perdeuteration drastically limits the structural information available from the NOE. Fractional deuteration also has its own problems with respect to sensitivity and its limited applicability as a general solution to the dipolar broadening displayed by proteins above 35 kDa. Spectroscopic solutions are also appearing. Some find their roots in the steady improvement in the use of the rotating frame to provide for more efficient isotropic mixing for coherence transfer. One very recent advance is the selection of the narrow multiplet component arising due to the (fortuitous) cancellation of dipole--dipole coupling and chemical shift anisotropy in .sup.15 N--.sup.1 H correlation experiments (Pervushin et al., 1997, Proc. Natl. Acad. Sci., USA, 94:12366-12371). This particular approach will not relieve the limitations in other contexts. In short, though these and other current approaches are extremely helpful, they do not appear to be generally applicable nor generally robust.
Reverse Micelle Technology
Reverse micelles form spontaneously as transparent solutions in a low polarity liquid and are thermodynamically stable assemblies of surfactant molecules organized around a water core. Reverse micelles were the subject of extensive attention in the 1980s as potential devices for a range of applications including separations, chromatography and reaction processes (Goklen & Hatton, 1985, Biotechnology Progress, 1:69-74). More recently, they have become the focus of further attention in the context of hosting various chemical reactions in solvents with low environmental impact such as supercritical carbon dioxide (Johnston et al., 1996, Science 271:624-626).
The size and stability of reverse micelles is dependent upon the amount of water loading. Water loadings have been described that yield stable reverse micelles of AOT in a variety of long and short chain alkanes large enough to accommodate proteins (e.g., Frank & Zografi, 1969, J. Colloid Interface Sci. 29:27-35; Gale et al., 1987, J. Am. Chem. Soc. 109:920-921; Fulton & Smith, 1988, J. Phys. Chem. 92:2903-2907; Fulton et al., 1989, J. Phys. Chem, 93:4128-4204).
For the analysis of protein structure, while solid state NMR methods continue to show great progress and recent successes like the determination of the gramicidin channel illustrate the potential of these approaches (Ketchem et al., 1996, J. of Bimolecular NMR 8:1-14), solution NMR methods are easier to employ. However, the difficulty of dealing comprehensively with large proteins in a general manner remains as a significant limitation to applying solution NMR methods to the rapidly growing list of proteins being discovered by the molecular biology community.
Thus, there is an ongoing need for novel techniques and approaches for extending the technique of solution NMR to proteins, especially, larger proteins, and other macromolecules. For example, fully 25% of known open reading frame sequences appear to code for membrane proteins and over 50% code for proteins that are beyond the size accessible by current solution NMR methods.